Thursday, November 21, 2024
11/21/2024
ABSTRACT Aspergillus fumigatus (A. fumigatus) is an important human fungal pathogen which is responsible for significant morbidity and mortality, particularly among immunocompromised patients. In recent years, we have made important progress in understanding the molecular mechanisms that regulate sensing of pathogens such as A. fumigatus by pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), and AIM2-like receptors (ALRs). Certain members of the NLR and ALR family assemble a cytoplasmic multi-protein complex termed the ‘inflammasome’, which activates caspase-1 and induces maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18 and drives pyroptotic cell death. The inflammasome plays critical roles in clearing fungal infections. My laboratory is a founding member of the inflammasome field and continues to radically advance this research area with our pivotal studies, elucidating the mechanisms driving inflammasome activation and identifying the importance of these pathways in immune responses. Our strong track record of studies focused on inflammasomes and pyroptosis has also allowed us to also make important discoveries about programmed cell death pathways. We fundamentally advanced the field of cell death by identifying extensive molecular crosstalk among seemingly distinct pathways. For example, we characterized Z-DNA-binding protein 1 (ZBP1) as an innate sensor of influenza A virus that triggers the NLRP3 inflammasome and pyroptosis as well as apoptosis and necroptosis (PANoptosis). We also identified that PANoptosis is mediated by a complex called the PANoptosome, which provides a molecular scaffold that allows interactions and activation of the machinery required for the inflammasome/pyroptosis, apoptosis, and necroptosis. As a result of the significant progress made during the last funding period (which resulted in 129 peer-reviewed papers since the submission of this R37 5 years ago, including publications in Cell, Nature, Nature Microbiology, Nature Immunology, and Immunity), we are now in an excellent position to pursue new directions that will investigate the molecular, biochemical, and cellular mechanism of inflammasome activation and PANoptosis in response to the global pathogen A. fumigatus. Our recent studies demonstrated that the novel A. fumigatus PAMP galactosaminogalactan (GAG) activates the NLRP3 inflammasome to drive cell death and in vivo protection against fungal-induced mortality. In addition to inflammasome activation, we also recently discovered that A. fumigatus induces PANoptosis and that the innate immune sensor ZBP1 is critical for driving inflammatory cell death. However, the molecular mechanisms and signaling cascades that regulate and control these pathways, along with the implications of PANoptosis in vivo, remain unknown. In this grant application for extension of our R37, we propose to investigate the signaling pathways regulating formation and activation of PANoptosomes driven by A. fumigatus infection. We will use a multifaceted approach, employing cellular, biochemical, genetic, and unbiased large-scale screening experiments, to identify key regulators and components of the PANoptosome and elucidate the fundamental molecular mechanisms of PANoptosis. We will employ a CRISPR-based whole-genome functional screening approach to determine which genes are essential for PANoptosis and to characterize their functions. We will treat a pool of cells in which each non-essential gene in the genome is deleted in individual cells with A. fumigatus-derived PANoptosis triggers and positive select for cells that are deficient in key PANoptotic components. We will then perform next-generation sequencing to identify the disrupted genes. The power of this system presents a tremendous opportunity to reveal novel sensors and regulators in PANoptosis. Furthermore, we will also perform immunoprecipitations of the PANoptosome components and incorporate targeted and unbiased proteomic approaches including mass spectrometry-based analyses and computational tools to identify and characterize the full spectrum of PANoptosis components upon infection with A. fumigatus. In addition to using molecular techniques to characterize the newly identified PANoptotic regulators, sensors, and componenets, we will also analyze where in the PANoptotic pathway each of these molecules functions using genetic approaches. We have developed advanced tools to study innate immunity, generating numerous knockout mouse strains that will be critically important for evaluating the newly identified regulators and members of the PANoptosome and PANoptotic pathway. As we have done with the PANoptotic regulators previously identified in our laboratory, such as ZBP1, we will use these established models to systematically dissect where these molecules fit into the pathway and rigorously test their roles in host defense against A. fumigatus infection. Continuation of this R37 and completion of our aims is expected to identify major innate immune signaling pathways and the molecular mechanisms that regulate the PANoptosome and PANoptosis in response to A. fumigatus. These discoveries are expected to identify novel signaling pathways that could be targeted by therapeutic interventions.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
